The broad long-term objective of this project is to develop Plasmodium vivax merozoite surface protein-3 (PvMSP-3) and PvMSP-9 recombinant proteins as candidate malaria vaccine products. This laboratory originally identified, expressed, and characterized these proteins and their potential as malaria vaccine candidates. The specific aims of the proposed studies are to: 1) Optimize the expression of PvMSP-3 and PvMSP-9 full-length and partial gene constructs in bacterial or yeast expression systems to obtain appropriate products for further testing of these antigens as malaria vaccine candidates; 2) Identify natural immunogenic B and T cell epitopes in PvMSP-3 and PvMSP-9 by evaluating currently available blood samples from individuals with different levels of exposure to P. vivax malaria infections in endemic areas of Brazil; 3) Evaluate immune responses in mice and rabbits, generated against several PvMSP-3 and PvMSP-9 candidate vaccine containing B and T cell epitopes formulated in clinical grade adjuvants; and 4) Test the safety, immunogenicity and efficacy of selected PvMSP-3 and PvMSP-9 candidate vaccine formulations in non-human primates. This research aims to produce up to three candidate products that can be evaluated for vaccine efficacy in New World monkeys. These primates are susceptible to P. vivax infections and can serve very well as a direct challenge model. The most promising products will also be tested in rhesus macaques for safety and immunogenicity in order to judge the potential for producing adequate responses in human clinical studies. Importantly, challenge with P. cynomolgi blood-stage parasites (the malaria species most closely related to P. vivax) will also be carried out in these immunized animals to determine if cross-species protection could be achieved. If warranted, to maximize the potential to evaluate protection in this model, the P. cynomolgi counterpart immunogens may also be produced and tested for efficacy and the ability of secondary infections to boost functional immunity in macaques. These combined approaches will help to identify the most efficacious PvMSP-3 and PvMSP-9 vaccine immunogens. This proposal will advance the development of several P. vivax vaccine candidates based on these antigens and also serve to establish reliable procedures to test and evaluate antigens in non-human primate models for safety, immunogenicity and efficacy.